Targeted and Repetitive Chromosomal Integration Enables High-Level Heterologous Gene Expression in .

is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with the Cre/ site-specific recombination system, with an efficiency of ∼3.7 × 10 CFU/µg DNA. A gene was successfully integrated into six selected chromosomal sites, and the relative fluorescence intensities (RFUs) of the resulting integrants varied up to ∼3.7-fold depending on the integrated site, among which the site integration showed the highest RFU. However, integrants with gene(s) integrated into the site showed various RFUs, ranging from 993 ± 89 to 7,289 ± 564 and corresponding to 1 to 13.68 ± 1.08 copies of gene integration. Moreover, the integrant with 13.68 ± 1.08 copies of the gene had a more stable RFU after 63 generations compared to that of a plasmid-engineered strain. To investigate the feasibility of this system for bioactive molecules with high expression levels, the fimbrial adhesin gene, , from was tested and successfully integrated into the site with 5.51 ± 0.25 copies, and the integrated achieved stable expression. All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location in Lactic acid bacteria (LAB), including , have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.